HBV P区聚合酶基因变异株的检测及限制性内切酶分析

Detection and RFLP analysis of the polymerase gene mutation of HBV DNA

目的 建立HBV聚合酶基因变异株DNA检测技术 ,探讨变异株的变异规律及其临床意义。方法 采用限制性内切酶酶切位点引入法设计引物检测YIDD、YVDD ,内切酶分析 ,PAGE电泳法检测DMHD、LMLL、LMAQ基因变异。结果 应用SSPⅠ及ApaL内切酶可准确地检测出YIDD和YVDD。 2 3例HBVDNA阳性病例中 ,9例YMDD未变异 ,BstNⅠ切点保持着野生型序列与参考序列相同 ;6例YMDD未变异 ,BstNⅠ切点发生变异 ;2例YMDD未变异 ,LMLL变异、BstNⅠ切点消失 ,并伴随有YAAV 4个氧基酸发生变异 ;1例YMDD未变异 ,DMHD发生变异 ;2例YIDD变异株伴随LMAQ变异、BstNⅠ切点变异 ;另 1例YIDD变异株在 50 2位 544位伴随YQHG、YKHG和SNLY、SHLY变异 ;3例YIDD和 2例YVDD病例的BstNⅠ切点均有变异。结论 研究结果发现 ,在YMDD未变异株中 50 % ( 9/1 8)为野生株 ,50 % ( 9/1 8)有基因变异 ,提示LMAQ、YIDD、YVDD变异 (W 2～W 1 2 )

Objective To develop a method to detect the polymerase gene mutation of HBV DNA and to explore the rule and clinical significance of mutant.Methods The primers were designed by induction of restriction endonuclease site to detecte YIDD and YVDD.Results YIDD and YVDD mutant can be accurately detected by S sp Ⅰ and A pa L I restricted enzyme.In 23 HBV DNA possitive patients ,9 had no mutation of YMDD and Bst N Ⅰ site kept the sequence of wild type and same with the reference sequence,6 had Bst N Ⅰ site mutant but no YMDD mutant,2 samples had LMLL mutant, Bst N Ⅰ site disappearing,no YMDD mutant and accompanyied by 4 amino acid mutant of YAAV.One samples had DMHD mutant but no YMDD mutant.2 YIDD mutants was accompanied by mutation of LMAQ and Bst N Ⅰ site mutant.Another YIDD mutant was accompanyied by YQHG,YKHG and SNLY SHLY mutant at 502 and 544 site.3 YIDD mutants and 2 YVDD mutants had mutation at Bst N Ⅰ site.Conclusion The results indicated that among the YMDD unmutant 50% are wide type but the others 50% have gene mutant.